Protamine (PRT) sulfate is the only commercially approved therapy for the reversal of heparin anticoagulation after cardiopulmonary bypass (CPB). Complications of PRT in CPB range from mild symptoms (hypotension) to rare life-threatening side-effects (acute pulmonary hypertension and/or anaphylaxis). In the course of studying immune complications of heparin (H), we have recently described a new and previously uncharacterized complication of protamine therapy, development of PRT/H antibodies (Abs). In preliminary studies, we show that ~40% of patients undergoing CPB develop PRT/H antibodies with minimal cross-reactivity to other heparin binding proteins, such as platelet factor 4 (PF4). Based on these observations, we hypothesize that PRT/H antibodies occur frequently after CPB and are associated with adverse clinical outcomes. To test this hypothesis, we will employ the R21 exploratory mechanism to investigate the clinical significance of PRT/H Abs in CPB patients. Specifically, we will: 1) Establish the incidence of PRT/H antibodies and investigate PRT/H antibody-associated clinical outcomes in a recently completed study of patients undergoing CPB (HIT 5801 Study). The HIT 5801 study is a recently completed study of ~1000 patients undergoing CPB in whom clinical data and plasma samples are available for further investigation. We have received IRB approval to examine the Duke cohort (n=783) for development of PRT/H antibodies and associated clinical outcomes. We will also establish the concurrent expression of PF4/H antibodies (causative antibodies in Heparin-Induced Thrombocytopenia or HIT) and determine if patients with PRT/H and PF4/H antibodies have worse outcomes. 2) Define the biologic characteristics of anti-PRT/H antibodies using in vitro and in vivo studies. Our preliminary studies indicate that the immune response to PRT/H and PF4/H share several biologic features. In this aim, we will examine the serologic and functional characteristics of PRT/H antibodies with respect to platelet and monocyte activation. In additional studies, we will utilize an existing rodent CPB model to study risk factors for and pathogenicity of PRT/H antibodies levels. Preliminary data obtained from this R21 funded study will allow us to document the clinical relevance of PRT/H Antibodies in CPB and enable future prospective investigations of outcomes related to antibody formation.